【作者】 杨联河；

【导师】 赵瑾；

【作者基本信息】 河南大学 ， 高分子化学与物理， 2011， 博士

【摘要】 化疗是治疗肿瘤的重要手段之一。化疗药物的主要缺陷是缺乏肿瘤细胞选择性。将药效团与多胺基团结合,形成多胺缀合物,可提高药效团与肿瘤细胞的亲和力,进而具有肿瘤细胞选择性。萘酰亚胺及其衍生物是DNA嵌入剂,具有显著的抗肿瘤活性。将萘酰亚胺与多胺缀合,可增加萘酰亚胺的水溶性和抗肿瘤活性,并使之具有肿瘤细胞选择性。因此,开发具有靶向性转运萘酰亚胺进入肿瘤细胞的萘酰亚胺-多胺缀合物是当今化疗研究领域中一个新的热点。然而,对萘酰亚胺-多胺缀合物抗肿瘤机制的研究尚不广泛,其机制尚未完全澄清。本文着重探讨单萘酰亚胺-亚精胺（mononaphthalimide spermidine, MNISpd）缀合物的多胺转运通道（polyamine transport, PAT）识别性和靶向性,对人宫颈癌HeLa细胞活力影响的机制,及其与活性氧（reactive oxygen species, ROS）间的关系。本文实验表明,在48h内,MNISpd对HeLa细胞的IC₅₀为4.71±0.21,对人胚肺成纤维细胞HELF的IC₅₀为16.68±0.18,对HELF细胞的细胞毒作用显著小于对HeLa细胞的细胞毒作用。MNISpd与SPD联用对HeLa细胞的IC₅₀为7.37±0.16,提示,SPD对HeLa细胞具有保护作用;MNISpd与DFMO联用对HeLa细胞的IC₅₀为3.47±0.05,提示,DFMO对MNISpd具有增效作用。以MNISpd<6μM处理HeLa细胞可诱导其细胞周期阻滞于S期,导致其增殖抑制。MNISpd在此浓度范围内诱导的HeLa细胞周期阻滞与p21表达活性上调、Cdc2表达活性下调有关,而与CDK2的表达无关。以MNISpd≥6μM处理HeLa细胞,可诱导其凋亡,且在48h内,9μM的MNISpd最适合诱导HeLa细胞凋亡。实验进一步表明,MNISpd介导的HeLa细胞凋亡与XIAP表达活性下调、caspase-3和caspase-9表达活性上调、线粒体膜电位去极化及继发的线粒体释放细胞色素c（cytochrome c）、凋亡诱导因子（apoptosis inducing factor, AIF）从线粒体中转移至细胞核有关,但对caspase-8的表达活性没有影响。同时,MNISpd介导的HeLa细胞凋亡与Bax表达活性上调、Bcl-2表达活性下调有关。当以10mM的N-乙酰半胱氨酸（N-acetyl-cysteine, NAC）预孵育HeLa细胞2h时,MNISpd介导的诱导HeLa细胞凋亡的事件均被抑制,但细胞周期仍阻滞于S期。MNISpd介导的活性氧在HeLa细胞的蓄积与其PAO活性上调、谷胱甘肽含量的复杂性变化有关。基于以上实验结果,本文认为,①MNISpd能被PAT识别,并通过PAT进入HeLa细胞;②MNISpd对HeLa细胞具有靶向性;③MNISpd对HeLa细胞活性的抑制主要源于诱导细胞凋亡和增殖抑制,且与MNISpd的处理浓度有关:在MNISpd<6μM,以诱导细胞增殖抑制为主,在MNISpd≥9μM,以诱导细胞凋亡为主;④MNISpd诱导的HeLa细胞凋亡可通过caspase依赖性内源性途径和AIF介导的非caspase依赖性途径发生,且与caspase依赖性外源性途径无关;⑤MNISpd诱导的HeLa细胞凋亡与MNISpd介导的氧化应激有关;MNISpd介导的活性氧在HeLa细胞的蓄积与还原性谷胱甘肽代谢池的耗竭有关,且PAO活性上调可能是ROS产生的源泉;⑥MNISpd诱导的HeLa细胞凋亡被NAC所抑制而其诱导的细胞周期阻滞则不受NAC预孵育的影响。另外,本文实验还表明,MNISpd可诱导人白血病K562细胞的增殖抑制和细胞凋亡。综上所述,MNISpd可以作为靶向性抗肿瘤药物进一步开发。更多还原

【Abstract】 Chemotherapy is one of the important developing strategies in cancer therapy. The major shortcoming of current cancer chemotherapies of many anti-cancer drugs is poor selectivity between tumor cells and healthy cells. Conjugating an anti-tumor agent with a polyamine motif elevates their affinity for tumor cells, and expresses excellent selectivity between tumor cells and normal cells. Naphthalimides and their derivates exhibit considerable potential as DNA intercalating anti-tumor compounds. Conjugating naphthalimides with a polyamine motif enhances their aqueous solubility and anti-tumor effects. Naphthalimide-polyamine conjugates are capable of specific entry into tumor cells. Then, developing naphthalimide- polyamine conjugates having the potential of transporting naphthalimide selectively into tumor cells is attractive in improving chemotherapeutic efficacy. However, the evaluation of their cytotoxic mechanism has not been comprehensive, and exact cytotoxic mechanism of these conjugates is not well known.This study focused on the effects of mononaphthalimide spermidine （MNISpd） conjugate on recognization by polyamine transport （PAT）, selectivity between tumor cells and healthy cells, growth and survival, and the relationship between MNISpd-induced anti-tumor effects and reactive oxygen species （ROS） in HeLa cells. Our results determined that after 48 h, an IC₅₀ of 4.71±0.21 and 16.68±0.18 was observed for HeLa cells and HELF cells respectively, and MNISpd exerted a higher cytotoxicity towards HeLa cells but with less cytotoxicity on HELF cells. An IC₅₀ of 7.37±0.16 and 3.47±0.05 was observed for HeLa cells treated with MNISpd plus SPD and MNISpd plus DFMO for 48 h respectively, indicating a protection effects for SPD in MNISpd plus SPD treatment HeLa cells and a synergism effects for DFMO in MNISpd plus DFMO treatment HeLa cells. In characterizing the mechanism of MNISpd cytotoxicity in HeLa cells, inhibition of proliferation was observed when HeLa cells were treated with MNISpd < 6μM and MNISpd induced a cell cycle arrest in S phase correlated with enhanced p21 expression and decreased Cdc2 but not CDK2 expression in this range. There were evidences of apoptosis in MNISpd≥6μM treatments HeLa cells, and 9μM MNISpd was suitable to induce apoptosis during a 48-h period. Our findings further determined that MNISpd-induced apoptosis was correlated with decreased XIAP expression and a loss of mitochondrial membrane potential following cytochrome c release, elevation of caspase-3, -9 activity, apoptosis inducing factor （AIF） translocation and up-/down-regulation of Bax/Bcl-2 protein expression, but not caspase-8, and these effects were completely antagonized by pre-incubation with 10 mM NAC for 2 h. MNISpd induced a significant ROS accumulation following up-regulation in polyamine oxidase （PAO） activity and complex variations in glutathione levels. Based on all these evidences, it is proposed: （1） MNISpd could be recognized by PAT and transfer naphthalimide entry into HeLa cells via PAT, （2） MNISpd expressed a targeting effect to HeLa cells, （3） MNISpd-mediated cytotoxicity towards HeLa cells primarily results from apoptosis induction and proliferation inhibition dependent on variations: with MNISpd < 6μM treatment, effects on cell proliferation predominate, while MNISpd≥9μM, apoptosis develops, （4） MNISpd-induced apoptosis via both intrinsic caspase-dependent and AIF-mediated caspase independent apoptosis pathways, but not extrinsic caspase-dependent pathway in HeLa cells is related to oxidative stress, （5） MNISpd-induced ROS production results from GSH （reduced form of glutathione） pool depletion, and PAO is likely to be the source of ROS, （6） MNISpd- induced apoptosis susceptible to be inhibited with NAC treatment but not cell cycle arrest in HeLa cells.In addition, MNISpd-mediated cytotoxicity towards k562 cells was related to apoptosis induction and proliferation arrest.It is concluded that MNISpd could act as potential tumor targeting agent for further development.更多还原